1. Field of the Invention
This invention relates to a gene, the GR gene, cloned from tomato mutants, a construct containing the gene and its promoter, a vector and method of transforming plants utilizing the construct and vector, and plants transformed with the gene construct.
2. Description of the Relevant Art
The ripe phenotype is the summation of biochemical and physiological changes occurring at the terminal stage of fruit development rendering the organ edible and desirable to seed dispersing animals and valuable to humans as an important food source and an agricultural commodity. Ripening changes, although variable among species, generally include modification of cell wall ultrastructure and texture, conversion of starch to sugars, increased susceptibility to post-harvest pathogens, alterations in pigment biosynthesis/accumulation, and heightened levels of flavor and aromatic volatiles (Hobson and Grierson. 1993. In: Biochemistry of Fruit Ripening, Seymour et al., eds., Chapman and Hall, London, pp. 405-442). Several of these ripening attributes translate to decreased shelf life and high input harvest, shipping and storage practices, particularly via changes in firmness and the overall decrease in resistance to microbial infection of ripe fruit. Currently acceptable techniques for minimizing the consequences of undesirable ripening characteristics include premature harvest, controlled atmosphere storage, pesticide application, and chemically induced ripening to synchronize the time of maturation. Unfortunately, added production, shipping and processing expenses, in addition to reduced fruit quality, are often the consequence of these practices, challenging the competitiveness, quality, and long-term sustainability of current levels of crop production.
The regulatory pathways that control fruit ripening are not fully understood although comparative analysis indicates that there is an emerging theme of conservation. For example, in silico mining of EST collections has revealed conservation of transcription factors that show ripening-related expression in tomato, a climacteric fruit, and grape, a non-climacteric fruit (Fei et al. 2004. Plant J. 40: 47-59). In addition, the RIN gene that encodes a MADS box protein essential for ripening in tomato (Vrebalov et al. 2002.) is functionally conserved in melon and strawberry (Binzel et al., unpublished data; Manning et al., unpublished data). In climacteric fruit that includes tomato, banana, apples and stone fruits, there is a conserved increase in respiration and ethylene synthesis that occurs at the onset of ripening (Lelievre et al. 1997. Physiol. Plant. 101: 727-739). The importance of ethylene for the co-ordination and completion of ripening in climacteric fruit has, been demonstrated through treatment of fruit with inhibitors of ethylene synthesis and action (Hobson et al. 1984. J. Plant Physiol. 116: 21-30; Yang, S. F. 1985. Hortscience 20: 41-45) and in transgenic and mutant plants blocked in their ability to produce or respond to ethylene (Klee et al. 1991. Plant Cell 3: 1187-1194; Oeller et al. 1991. Science 254: 437-439; Picton et al. 1993. Plant J. 3: 469-481; Wilkinson et al. 1995. Science 270: 1807-1809)
Altered ethylene responsiveness in plant tissues affects normal development and can compromise the plants ability to respond to environmental stimuli (Bleeker et al. 1988. Science 241: 1086-1089; Guzman and Ecker. 1990. Plant Cell 2: 513-524; Lanahan et al. 1994. Plant Cell 6: 521-530; Wang et al. 2002. Plant Cell 14: S131-S151). The mechanisms by which the ethylene signal is perceived and transduced to mediate phenotypic changes is not fully understood although many elegant studies exploiting the triple response screen in Arabidopsis have led to the identification of critical components of this signaling pathway (Guo and Ecker. 2004. Curr. Opin. Plant Biol. 7: 40-49).
The ethylene signal is initially perceived by a family of receptors that share homology to bacterial two-component regulators (Chang et al. 1993. Science 262: 539-544; Hua et al. 1995. Science 269: 1712-1714; Hua et al. 1998. Plant Cell 10: 1321-1332; Sakai et al. 1998. Proc. Natl. Acad. Sci. USA 95: 5812-5817). Loss of function analysis indicates that the receptors act in a semi-redundant manner to negatively regulate ethylene responses (Hua and Meyerowitz. 1998. Cell 94: 261-271). At least two receptors interact with Constitutive Triple Response 1 (CTR1), a serine threonine MAPKKK that acts as a negative regulator of the pathway (Kieber et al. 1993. Cell 72: 427-441; Clark et al. 1998. Proc. Natl. Acad. Sci. USA 95: 5401-5406; Gao et al. 2003. J. Biol. Chem. 278: 34725-34732). An integral membrane protein, EIN2, with homology to the NRAMP family of metal ion transporters acts downstream of the receptors and CTR1 (Roman et al. 1995. Genetics 139:1393-1409). The biochemical function of EIN2 remains unknown but genetic studies have indicated that all ethylene responses described to date are transduced through this signaling intermediate (Hall and Bleecker. 2003. Plant Cell 15: 2032-2041). A family of transcription factors encoded by EIN3 and EIL (EIN3-like) act downstream of EIN2 (Chao et al. 1997. Cell 89: 1133-1144; Solano et al. 1998. Genes Dev. 12: 3703-3714). Homodimers of EIN3, EIL1 and EIL2 bind to a defined target site in the promoter region of the transcription factor, Ethylene Response Factor 1 (ERF-1) (Solano et al., supra). ERF1 is part of a large multigene family of transcription factors and is important in the regulation of downstream ethylene responsive genes via binding to the “GCC” box promoter element (Ohme-Takagi et al. 2000. Plant Cell Physiol. 41: 1187-1192; Fujimoto et al. 2000. Plant Cell 12: 393-404). Ethylene responses are regulated at the level of EIN3 via ubiquitin/proteasome-dependent proteolysis mediated by the F-box proteins, EBF1 and EBF2 (Guo and Ecker. 2003. Cell 115: 667-677; Potuschak et al. 2003. Cell 115: 679-689).
The importance of ethylene in regulating traits of agronomic importance, particularly fruit ripening and floral senescence, has driven research on the identification and functional characterization of components of the ethylene signaling pathway in crop species (Klee, H.-J. 2004. Plant Physiol. 135: 660-667; Adams-Phillips et al. 2004a. Trends in Plant Science 9: 331-338). Studies utilizing tomato and petunia have been at the forefront of this comparative analysis and have revealed structural and functional conservation of the ethylene signaling pathway (Adams-Phillips et al. 2004b. Plant Mol. Biol. 54: 387-404; Leclercq et al. 2002. Plant Physiol. 130: 1132-1142; Shibuya et al. 2004. Plant Physiol. 136: 2900-2912; Tieman et al. 2001. Plant J. 26: 47-58; Tieman et al. 2000. Proc. Natl. Acad. Sci. USA 97: 5663-5668; Wilkinson et al. 1995. Science 270: 1807-1809). Interestingly there is an expansion of the gene families encoding the receptors and CTR components in tomato and other crop plants adding a further layer of complexity to the ethylene response pathway (Klee, H.-J., supra; Adams-Phillips et al. 2004a, b, supra). Expression studies of these genes further suggest tissue-specific transcription of some receptors (Tieman and Klee. 1999. Plant Physiol. 120: 165-172), though no ethylene signaling genes that function exclusively or even predominantly in fruit or related floral tissues have been described to date.
Thus, the plant hormone ethylene has profound effects on fruit ripening and senescence, conditions that ultimately result in a deterioration of quality in a wide range of horticultural crops. Having greater understanding of how ethylene regulates ripening and senescence provides us with tools for improving agricultural production as well as products with enhanced nutritional and flavor attributes.
This invention concerns the cloning of a novel gene, GR, at the Gr/Nr-2 locus that is able to differentially regulate tissue-specific ethylene responses in tomato with the most dramatic effect observed during inhibition of fruit ripening.